Nematodes In Tomato Crops Biology Essay

Chapter 1INTRODUCTION

Tomato ( Lycopersicon lycopersicum Karst ) belonging to household Solanaceae is the most popular and widely consumed vegetable harvest ( Khoso, 1988 ) . Out of 15 veggies, tomato ranks at 2nd in footings of entire universe one-year production. In Pakistan it is cultivated over 47.

10 1000s hectares with the one-year production of about 50.23 1000s metric tons ( MINFAL, 2007 ) . It is grown all over Pakistan in different seasons as the climatic and adaphic conditions of Pakistan are favourable for bring forthing high quality tomatoes ( Chaudhary, et al. , 1995 ) .Historical groundss showed that tomato is of Tropical American beginning and said to be foremost domesticated in Mexico.

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From Tropical America, it was so introduced in Europe, West Africa, Tropical Asia and throughout the tropical parts all over the universe. Whatever may be its early history of cultivation, the tomato has got enormous popularity from the center of nineteenth century to the present clip ( Tiwari and Choudhury, 1986 ) . In Pakistan, two harvests of tomato are produced yearly, foremost in spring and 2nd in fall. However, in most countries of Sindh tomatoes can be grown throughout the twelvemonth.

It is the major vegetable hard currency harvest in Sindh, Punjab and Balochistan ( Badshah el al. , 1997 ) .As compared to other veggies, it is rich in nutritionary value and available relatively at low monetary values. In every place tomato is used in different ways like salad, catsup, chatni and in other delightful dishes. Proximate composing of tomato fruit shows that 100 gms of comestible part contains 93.76 g of H2O, 0.

85 g protein, 0.33 g entire lipoids, 4.64 g saccharides, 1.1 g entire dietetic fibre and 0.42 g ash. A fresh altogether tomato besides contains an exceeding sum of vitamin A ( 623IU/100 g ) and B1/Thiamin ( 0.059 mg/100 g ) . It is an first-class beginning of vitamin C/ascorbic acid ( 19.

1-23 mg/100 g ) , which is normally more lacking in our diet as comparison to vitamin A or B. in add-on, it contains Riboflavin ( 0.048 mg/100 g ) and Niacin ( 0.628 mg/100 g ) .100 g of comestible part of tomato besides contains Mg ( 11 milligram ) , P ( 24 milligram ) , Na ( 9 milligram ) , Fe ( 0.

45 milligram ) , K ( 222 milligram ) and Ca ( 5 milligram ) . Tomato is said dieter dream in its natural province being really low in thermal fruit incorporating 21 kcal/88 kJ.In add-on to nutritionary value, tomato fruit act as nutraceutical holding high medicative value. Its mush and juice act as digestive stimulation, mild laxative, a booster of stomachic secernment and blood purifier. It is besides considered to be enteric antiseptic and utile in malignant neoplastic disease of oral cavity and rancid oral cavity.

It stimulates inert liver and is good in chronic indigestion ( Tiwari and Choudhury, 1986 ) . Tomato is a major beginning of lycopene, which act as anti-oxidant and anti-cancer agent. Tomatos are linked in peculiar to take down rates of pancreatic malignant neoplastic disease and cervical malignant neoplastic disease. It appears that the ingestion of tomatoes ( high in vitamin C ) confers benefits in forestalling and handling malignant neoplastic disease.

The mean output of tomato in Pakistan is really low as compared to other tomato turning states of the universe. There can be several grounds for this low output such as altering climes conditions, conventional methods of cultivation, uncertified low giving up assortments and biotic factors. The biotic factors that affect the productiveness and quality of tomato are fungi, bacteriums, viruses, roundworms, weeds and insects. Among these biotic factors root knot roundworms are known to do terrible harm to tomato in different parts of the universe ( Dropkin, 1980 ) and tomato is considered as the most favourable host for root-knot roundworms ( Lamberti, 1979 ) .Nematodes, belonging to kingdom Animalia, are wormlike in visual aspect, holding broad host scope and bring on a assortment of diseases.

Nematodes are the most legion multicellular animate beings on Earth ( Kimpinski and Sturz, 2003 ) . Several 100 species of roundworms feed on life workss, obtain their nutrient with lances or stylets and do a assortment of works diseases. Nematodes cause 8 to 20 % losingss to major harvests, fruits and veggies worldwide, for a sum of over $ 87 billion yearly ( Sasser and Freckman, 1987 ) .Among the roundworms, doing different works diseases, root knot roundworms ( Meloidogyne spp. ) are the most annihilating and detrimental. These roundworms cut down universe harvest production by about 5 % .

Root knot nematode belongs to order Tylenchida, sub-order Tylenchina, ace household Heteroidea, household Meloidogynidae and genus Meloidogyne. Amongst all the roundworm genera, Meloidogyne genus ranked first and considered really of import works pathogen ( Dropkin, 1989 ) .The root knot roundworms are sedentary entoparasites and are among the most destructive agricultural plagues assailing a wide scope of harvests and do high degrees of economic losingss ( Mai and Abawi, 1987 ) .

They are found in tropical and semitropical countries of the universe and infect more than 2000 works species including about all cultivated workss ( Hussey, 1985 ) . More than 60 species of root-knot roundworms have been reported so far but M. incognita, M. javanica, M. Arenaria and M. hapla are comparatively more destructive accounting for 95 per centum of all root-knot nematode infestations in field harvests. The proportion of Meloidogyne incognita in the agribusiness soils is 52 % , M.

javanica 31 % , M. arenaria 8 % , M. hapla 7 % and other species are about 27 % ( Hussey and Janssen, 2002 ) .Among the species, M. incognita is widely prevailing, common and detrimental 102 works species in Pakistan.

The infection starts when 2nd phase juveniles ( J2 ) penetrate in roots. These roundworms set up feeding sites within the roots where they induce roots saddle sores or knots. Deprive workss of foods and cause cellular, metabolic and structural alterations within works tissues. These physiological and physical alterations to the works can cut down harvest output and quality drastically.

Nematodes assailing susceptible workss at seedling phase cause heavy losingss and may ensue in complete devastation of the harvest. But infections of older workss may demo merely minor effects on output or may cut down outputs perceptibly ( Agrios, 2005 ) .It is estimated that 24-38 % tomato workss are affected by these roundworms. The grade of harm caused by roundworms additions in combination with other dirt borne pathogens. Tomato workss can be killed when Fusarium oxysporum f. sp. lycopersici is present with Meloidogyne spp.

( Sikora and Fernandez, 2005 ) . The equal control of these biotic factors is straight related to dependable productiveness and good quality tomato fruit ( Csizinsky et al. , 2005 ) .Root knot roundworms are really hard to get by with because of their high generative potency and polyphagous nature. In order to avoid the subsequent addition of detrimental populations, 99.

9 per centum control should be ensured ( Whitehead, 1998 ) . The losingss due to root knot roundworms can be reduced by dirt fumigation, application of nematicides and by cultivating immune or non-host workss. But the conventional usage of chemical control is doing many terrible effects upon the human wellness every bit good as on the environment therefore the development of unconventional and environment friendly command methods are of great importance, the biological control is one of them.

There are more than 200 species of biological control agents, which largely include Fungis, viruses, bacteriums and predatory invertebrates ( Dasgupta, 1998 ) .Many compounds have now been withdrawn from usage, advancing the demand for new, safe and effectual options ( Zuckerman and Esnard, 1994 ) . Sing all the restraints in sight, the environmental effects, cost of chemicals and wellness jeopardies, present survey was carried out to prove the efficaciousness of biological control agents Trichoderma harzianum and Azadirachta indica against Meloidogyne incognita on tomato.The chief aim of current survey was:To analyze the person and combined consequence of T. harzianum and A. indica on M. incognita

Chapter 2 REVIEW OF LITRATURE

Root-knot roundworms are most of import and detrimental plagues holding broad host scope. They cause immense economic losingss to about all veggies ( Back et al.

, 2002 ) . So, root knot roundworms are needed to command. A great accent has been given on biological control because of its effectivity and environmental safeness. A brief reappraisal of biological control of root knot roundworms is given below.

Biological control by Fungi

Among the biocontrol agents that parasitize or quarry on roundworms and lessen roundworm populations by their counter actions, Fungis hold really of import place and some of the Fungis have shown great possible as biocontrol agents. Fungi continuously demolish roundworms in about all types of dirts because of their changeless association with roundworms in the rhizosphere.

The most of import genera which trap or quarry roundworms include Paecilomyces, Verticillium, Arthrobotrys, Hirsutella, Drechmeria, Fusarium, Nematophthora and Monacrosporium. Applications of these Fungis have given outstanding consequences against roundworms ( Siddiqui and Irshad, 1996 ) .When different concentrations of T. harzianum were used root knot nematode infection and other parametric quantities were significantly decreased in a green house experiment. Nematode egg hatching degree decreased significantly after the incursion T. harzianum to nematode egg mass matrix ( Sahebani and Hadavi, 2008 ) . The potency of T. harzianum to command the root-knot roundworm M.

javanica and its ability to colonise eggs and second-stage juveniles was evaluated. The application of T. harzianum resulted in decrease of root galling and increased top fresh weight in nematode-infected tomatoes ( Sharon et al. , 2001 ) . Acremonium strictum and T. harzianum both entirely and in combination showed promising public presentation by bettering the wellness of the tomato works with a singular decrease in M.

incognita population ( Goswami et al. , 2008 ) .When the two corn loanblends were grown with and without M. Arenaria in T. harzianum and T. koningii treated dirt the shoot and root growing of both loanblends was significally enhanced and the reproduction of M. Arenaria on the susceptible corn loanblend was suppressed, whereas the root knot immune loanblend was non affected ( Windham et al.

, 1989 ) Paecilomyces lilacinus strain 251 was evaluated for its possible to command the root-knot roundworm ( M. incognita ) on tomato. Pre-planting dirt intervention reduced root galling by 66 % , figure of egg multitudes by 74 % and the concluding roundworm population in the roots by 71 % compared to the inoculated control in growing chamber ( Kiewnick and Sikora ) .Trichoderma harzianum and Trichoderma viride reduced the incidence and pathogenicity of Meloidogyne incognita on tomato in In vitro experiments. All the isolates were found effectual and caused mortality in second-stage juveniles ( J2 ) as compared with the control ( Al-Fattah et al. , 2007 ) . Amendment of the civilization filtrate of a T. harzianum to P.

fluorescens growing medium improved root-knot roundworm biocontrol in in vitro and greenhouse conditions. When the T. harzianum and Pseudomonas fluorescens medium was applied to unsterilised dirt great decrease in nematode population densenesss was observed in tomato roots ( Siddiqui and Shaukat 2004 ) .Trichoderma harzianum isolated from dirt and root samples of tomato collected from root knot nematode-infested Fieldss of different vicinities of Malakand and Swat ( NWFP ) was tested against Meloidogyne javanica in the in vitro. Isolates collected from Jabban and Shamozai ( Th1 and Th9 ) , were more aggressive against M. javanica. Different concentrations of civilization filtrates ( CF ) of T.

harzianum significantly inhibited egg hatching of the roundworms ( Khattak et al.,2008 ) .The application of Trichoderma harzianum, T. hamatum and T. koningii civilization filtrates significantly reduced the egg-masses of reniform and root-knot roundworms.

Trichoderma species inhibited the roundworm activity and motions in vitro. The consequence of civilization filtrate was more important on Meloidogyne javanica egg than on larvae. The toxic metabolites of Trichoderma species straight inhibited nematode incursion and developments ( Bokhari 2006 ) .

Biological control by Bacteria

When lyophilized cyanophyte pulverization was incorporated into potted Weld dirt at the rate of 0.2 % , 0.4 % , 0.6 % , 0.

8 % and 1.0 % ( w/w ) 5 yearss before tomato planting, reduced root galling and increased vegetive growing of tomato workss and root-mass production. At high doses 0.8 % and 1.0 % ( w/w ) root galling and nematode population decreased by 68.9 % and 97.6 % severally compared with control ( Khan et al.

, 2007 ) . When rice roots were treated with Bacillus megaterium 40 % decrease was observed in root knot nematode incursion and saddle sore formation as compared with control. B. megaterium colonisation of rice roots decreased migration of M.

graminicola to the root zone by about 60 % compared control. Hatching was reduced over 60 % when M. graminicola eggs were exposed to secondary metabolites of B. megaterium compared with eggs non exposed ( Padgham and Sikora, 2007 ) .Juveniles of M. javanica ( 500, 5,000 or 10,000 ) were added to the pots, amended with the preparations of Pasteuria penetrans, and dried nim trees bar and foliages, before seting 6-week old tomato seedlings. After 64 yearss tomatoes were sampled, afterward second and 3rd harvests were sown for 59 and 67 yearss severally without farther applications of P.

penetrans and nim tree. The root-galling was reduced significantly at the terminal of 3rd harvest in combined intervention of P. penetrans and neem bar and this intervention besides had the antic consequence on the growing of the tomato workss ( Javed et al. , 2008b ) .Plant growth-promoting rhizobacteria colonize the rhizosphere and promote works growing or protect workss against certain works pathogens. Different commercially available rhizobacterial inoculums ( Equity, BioYield, and AgBlend ) and RhizoVital were selected to prove the suppressiveness of root knot roundworm.

Treatment with each of the inoculums increased works growing, root weight and reduced root chafing significantly ( Burkett-Cadena et al. , 2008 ) .

Biological control by Algae and Nematopathogenic Nematodes

Consequence of nematopathogenic roundworms was measured root-knot roundworms infecting peanut and tomato. Three hundred Meloidogyne hapla eggs and 25 IJs/cm2 of Heterorhabditis bacteriophora were inoculated to peanut seedlings in growing chamber. Treatment of H. bacteriophora to peanut seedling significantly reduced invasion and egg recovery of M.

hapla as compared with control. In nursery Peanut seedlings were infested with 5000 M. hapla eggs and treated with 25 IJs/cm2 of Steinernema riobrave S.

feltiae, or H. bacteriophora 2 hebdomads earlier, 1 hebdomad earlier, at the same clip, 1 hebdomad after, or 2 hebdomads after. Pre- and post-infestation applications of S. feltiae suppressed M. hapla incursion in pre- and post-application but non egg production. Tomato workss were besides inoculated with 5000 M. incognita eggs and 25 IJs/cm2 of S.

glaseri or H. megidis applied at the same times in the nursery. High rate of S.

glaseri reduced egg production and low rate of S. glaseri suppressed M. incognita incursion into tomato roots ( Perez and Edwin, 2004 ) .The consequence of inoculant denseness and application clip of Mononchoides fortidens against Meloidogyne Arenaria on tomato workss, grown in pots incorporating 500g Weld dirt inoculated with 10 2nd phase juveniles/g dirt, was measured. Treatment of pots with different concentrations ( 0, 50, 100, 150, 200, and 250 per pot ) of M. fortidens 7 yearss before tomato seting significantly reduced root galling and the concluding population of M. Arenaria and improved vegetive growing of tomato workss and root-mass production, compared with control ( Khan and Kim, 2005 ) .

Before transfering tomato seedling, roots were dipped in different concentrations of civilization i¬?ltrate of Microcoleus vaginatus for 30 proceedingss. Root-dip intervention reduced the root galling, population of M. incognita and improved vegetive growing of workss as compared to command.

as the concentration of civilization i¬?ltrate was increased the benei¬?cial ei¬ˆect of root-dip intervention besides increased. Root galling and i¬?nal roundworm populations were reduced by 65.9 % and 97.5 % , severally when treated at the highest concentration ( Khan et al. , 2005 ) .

Biological control by Botanical Poisons

Dirt amendments with assorted preparations of Neem ( A. indica ) such as sawdust, leaf pulverization and oilseed bars decreased plant-parasitic roundworms significantly relative to command secret plans.

All interventions increased fresh and dry weights and the tallness and figure of cods on garbanzo workss ( Akhtar, 1998 ) . Second phase juveniles of M. incognita were exposed to 500 ppm of each works infusion for 24, 48 and 72 h.

foliage infusions of, Aristolochia bracteolate, Solenostemma argel and Ziziphus spina-christi and the seed infusions of Datura stramonium, Aregimone mexicana and A. indica caused high mortality rates upto 94-80 % after 72 H of exposure. For most of the infusions, the mortality rate increased with increasing exposure clip ( Elbadri et al. , 2008 ) .M.

incognita eggs were exposed to root infusions of Dharek, Neem, Castor and Datura. One hundred per centum suppression of egg hatching and larval mortality was observed standard root infusions of Neem and Dharek. Decrease of egg suppression and larval mortality was observed with an addition in the dilution of the infusions. Similarly juvenile mortality was besides increased with an addition in exposure clip ( Kayani et al. , 2001 ) .When AM-fungi was combined with different oil bars ( R. communis, Brassica campestris and A.

indica ) resulted in cut downing the galling and nematode generation therefore bettering the works growing and output. The best consequences sing, reduced root infection, nematode reproduction and works growing and output were obtained with the combination of AMF and R. communis oil bar ( Bharadwaj and Sharma, 2006 ) .Raising mycorrhizal seedlings of tomato in dirt amended with nim tree ( A. indica ) bar enhanced works vegetive growing and decreased root galling and concluding population of M. incognita signifacantly. ( Rao et al.

, 1995 ) . Exposure of Second phase juveniles of M. javanica to aqueous infusions of Neem ( foliages and bar ) at 2.

5 % , 5 % , and 10 % w/v and a refined merchandise, Aza at 0.1 % w/v. The 10 % infusions of Neem foliage and bar caused 35 % and 28 % mortality and 83 % and 85 % stationariness, respectivelyas compared to command. ( Javed et al. , 2008a ) .Soil application with petroleum and refined preparations of nim tree in protective and healing mode had important consequence on figure of egg multitudes.

Protective application of neem foliages and coat did non cut down the invasion of juveniles whereas aza at 0.1 % w/w did. On the other manus healing application significantly reduced the figure of egg multitudes and eggs per egg mass as compared with the control ( Javed et al. , 2007a ) . Five concentrations of H2O soluble infusions of Ocimum sanctum, Ricinus communis, Carica papaia, Tagetes patula and A. indica were filtered, added to petri dishes and infested with the eggs of M. incognita. O.

sanctum showed best consequences with no hatching within 48 H vs. 34.8 % in the control ( Bharadwaj and Sharma, 2007 ) .The aqueous infusions of nim tree, Castor and pagomia bars were evaluated as substrate for mass production of T. harzianum and in the direction of M. incognita in egg works.

Castor bar infusions at 10 % gave maximal growing of mycelial mat and spore production of T. harzianum compared with moderate growing in 10 % pagomia bar and 5 % Castor bar infusions. Application of works based preparations of T. harzianum was effectual in bring forthing vigorous seedlings with least root galling ( Rao et al. , 1998 ) .Exposure of M. incognita eggs to concentrations of root infusions of Siam weed, Neem, Castor bean and Lemon grass significantly reduced egg hatching and increased larval mortality. 100 % concentration of root infusions of Siam weed and Neem exhibited 100 % egg hatch suppression and larval mortality.

While with same conc. of root infusions of Castor bean and Lemon grass resulted in 93 and 95 % suppression of egg hatch and 62.1 and 75 % larval mortality severally. An addition in the dilution of all the infusions, egg suppression and larval mortality was decreased. Similarly juvenile mortality was besides increased with an addition in exposure clip ( Adegbite and Adesiyan, 2005 ) .A mixture of Haplophyllum and Plectranthus oils ( 1:1 ) showed high toxicity to M. javanica by killing all nematode juveniles and suppressing egg hatching at 12.

5 mg/ml concentration after 24 H exposure clip in vitro. Tomatoes grown in dirt amended with a combination ( 1:1 ) of the two oils developed fewer root saddle sores than those grown in dirt treated with higher doses of either oil In the green-house ( Onifade et al. , 2008 ) .Neem bar ( A. indica ) and a biocontrol fungus, T. harzianum either singly or in combination were evaluated for the direction of M. incognita on tomato.

They significantly increased tomato works growing and reduced the root galling. The concluding populations of M. incognita were observed in tomato seedlings transplanted in neem cake-amended dirt incorporated with T. harzianum. Increase in colonisation of T. harzianum on roots of tomato was besides observed in the above interventions which indicated favourable effects of neem cake amendment on the growing of T. harzianum ( Rao et al.

, 1997 ) .It is obvious from the above reappraisal that biological control agents like T. harzianum and A. indica have a great potency to cut down root knot roundworms and addition works growing significantly.

MATERIAL AND METHODS

Chapter 3Collection of root samples

Root samples demoing characteristic symptoms of root- knot roundworm were collected from M.

incognita infected tomato field. Rootss were carefully lifted with little spade up to 20-25 centimeter deepness from the root zone along with about 1 Kg of adhering dirt. These samples were put in polyethylene bags, moistened good to guarantee the equal wet for endurance of roundworms. Samples were stored in icebox at 4A° C until processed.

Isolation of Root-Knot Nematode from roots and dirt

Root-knot roundworms were isolated from infested roots and dirt by White-head and Hemming tray method ( White-head and Hemming, 1965 ) . In this method, the septic roots with egg multitudes were sliced and chopped after rinsing exhaustively under tap H2O.

The dirt and roots were kept in a tray lined with tissue paper holding sufficient sum of H2O. After 24 hours, H2O was decanted off in a beaker and allowed to settle for one hr. When the juveniles were settled, the surplus of H2O was drained out until approximately 100 milliliters remained.Isolation of root knot nematodes from dirt samples was done by Cobb ‘s decanting and screening method. In this method, 2-3 litters of H2O was added to a plastic bath holding 250-500 g of dirt sample, exhaustively assorted until all balls were broken up. Heavy dirt atoms, roots and stones sunk to bottom and were drained out from H2O by manus. The supernatant H2O was poured through the harsh screen ( 36- mesh ) into the 2nd pail.

A litre of H2O was added and above measure was repeated to acquire optimal figure of roundworms. The H2O suspension was stirred, allowed to settle and distribute the supernatant H2O from the 2nd pail gently through the all right screen ( 100-mesh ) and allowed it to run down the drain. The screen was washed with a soft watercourse of H2O to take all right atoms. The same procedure was repeated by utilizing all right mesh screen ( 275 and 325 all right mesh screen ) . Finally the stuff left on the 325-mesh screen was transferred into a 500 milliliter beaker. The suspension was allowed to settle down to the underside of beaker for about one hr.

Excessive H2O was drained off with the aid of syphon tubing and numeration of roundworms in 1 ml H2O was made in three replicates.

Designation of Meloidogyne spp.

Root knot roundworm species was identified as Meloidogyne incognita on the footing of perineal form. Root saddle sore with mature female was selected and placed in ticker glass with H2O. The root tissue was teased apart with forceps to take grownup females. the cuticle of the female was ruptured near the cervix and gently pushed the organic structure contents out.

The cuticle was placed in a bead of 45 % lactic acid on a petri dish. The posterior half of the organic structure was cut off, farther the posterior piece of the cuticle holding perineal forms to square and wholly take interior tissue by flexible bristle. The perineal pattern-bearing part was transferred to drop of glycerol on a microscope slide. The glass screen faux pas was gently placed and sealed with paraffin and labeled. The form was examined under a research microscope ( Eisenback et al.

, 1981 ) . Two species were dominant M. incognita and M. javanica. M. incognita was purified for the farther experiments.

Extraction of Meloidogyne incognita eggs

Root knot infected tomato roots were collected and cut into 1-2 centimeter sections. Rootss were shaken smartly ( manually ) in 200 milliliter of a 0.

5 to 1.o % NaOCl solution for 1-4 proceedingss. NaOCl solution was passed rapidly through a 200-mesh screen, nested over 500-mesh screens to roll up freed eggs.

A watercourse of cold H2O was passed through 500-mesh screen to take residuary NaOCl and rinse for several proceedingss. The staying roots were rinsed for several times to take more eggs and so collected in beaker by screening ( Hussey and Barker, 1973 ) .

Mass culturing of Meloidogyne incognita

Three hebdomads old tomato seedlings were transplanted into earthen pots filled with formol sterilized flaxen loam dirt. After one hebdomad of transplantation, 4-5 little holes ( 3-cm deep ) were made around each works and 1000-1500 newly hatched juveniles of root knot roundworms were inoculated. These holes were covered with dirt to forestall drying. Plants were kept in green house at temperature scope 22-35 C and watered on a regular basis.

Plants were harvested after two months to roll up juveniles.

Preparation and sterilisation of dirt

Sandy loam dirt was used for the experiment. Soil was decently assorted, air dried and sieved to clean rocks and dust. After screening, diluted formol ( 1:320 ) was poured on a little pile of dirt and was covered for seven yearss with polythene sheet in such a manner that the exhausts were confined beneath the fictile sheet.

Turning tomato seedlings

Tomato cultivar “ Money Maker ” susceptible to M. incognita, was raised in earthen pots incorporating sterilized flaxen loam dirt and allowed to turn for 30 yearss.

Transplanting tomato seedlings

Tomato seedlings grown in earthen pots were shifted to divide pots holding dirt amended with biocontrol agents. Seedlings were shifted from earthen to divide pots carefully and so adhering dirt was removed gently by manus and so planted in pots. Nursery seedlings were handled carefully to avoid any hurt.

Mass culturing of Trichoderma harzianum

T.

harzianum was cultured on Potato Dextose Agar ( PDA ) at ( 27EsCA± ) . After obtaining the pure civilization, biological control agent was grown on wheat seeds. Wheat seeds ( 250 g ) were soaked in H2O for 12 hours. The besotted seeds were surface dried with paper towel, placed in a 500 milliliter. flask and were sterilized in an sterilizer for 50 proceedingss at 15 pounds per square inch. For the readying of spore suspension of the counter Fungi, 5 milliliter. distilled H2O was added to the petridish incorporating 10 to 14 day-old fungous settlement. With the aid of a sterilised cringle, the surface of the fungous settlement was rubbed gently to do a spore suspension.

Then the sterilized wheat grains was inoculated with the spore suspension and placed in the brooder for 15 yearss at the room temperature. The flask was shaken every twenty-four hours to advance the unvarying growing of the Fungi. The Fungi started growing after 4-5 yearss. After 14 yearss of vaccination, there was sufficient fungous growing for field application. The biocontrol agents will so be mixed in sterilised dirt at the rate of 0, 4, 6, 8 and 10 gm / Kg of dirt.

Preparation of Neem preparations

Neem foliages were washed carefully to take dust and so kept in sunshine to dry. Aqueous infusions were prepared by soaking pulverization of dried Neem leaves for 24 H in sterilized distilled H2O and filtered through muslin fabric so to acquire clear infusion, filtered through filter paper.

This served as standard infusion. 2.5 % , 5 % , 10 % and 15 % preparations were made by adding H2O in standard infusion.

Evaluation of biological control agents for the control of M. incognita

3.11.1. Consequence of biocontrol agents on mobility and mortality of Meloidogyne incognita

For appraisal of mobility and mortality, I ml of nematode suspension incorporating 500 newly hatched juveniles were exposed to different concentrations of T.

harzianum ( 1:10 and 1:100 ) , A. indica ( 5 % and 10 % ) and combination T. harzianum and A. indica, in 5cm plastic home bases. Home plates were placed in brooder and after 24 and 48 H informations was recorded sing mobility and mortality.

3.11.2. Consequence of biocontrol agents on egg hatching of M. incognita

For egg suppression surveies egg multitudes of unvarying colour and size were picked from M. incognita infected roots of tomato and carefully placed in dilutions of fungous filtrate, Neem pull out both entirely and in combination. Entire juveniles were counted after 2, 4, 6, 8 and 10 yearss.

3.

11.3. Consequence of biocontrol agents both entirely and in combination on works growing parametric quantities

The consequence of biocontrol agents ( T. harzianum and A.

indica ) on M. incognita was evaluated separately and in combination. One month old seedlings of tomato curriculum vitae.

Money Maker were transplanted to dirty amended with T. harzianum ( 2, 4, 6 and 8 g/1kg dirt ) and A. indica ( 2.5 % , 5 % , 10 % and 15 % ) separately and in combination.

One hebdomad after organ transplant, the workss were inoculated with newly hatched 2nd phase juveniles of M. incognita. Each intervention was replicated five times. Inoculation of 2nd phase juveniles to H2O amended dirts was used as control. The pots were placed in green house at 25-32 °C. After eight hebdomads of vaccination, information was recorded sing works tallness, fresh shoot weight, dry shoot weight, fresh root weight and figure of saddle sores per works. The information was analyzed by utilizing statistical bundles ( Steel and Torrie, 1997 ) .

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